Gene 763 of phytopathogenic fungus Magnaporthe grisea and use thereof for identifying fungicidal compounds

ABSTRACT

The invention concerns a novel nucleic acid fragment of the genome of rice pathogenic fungus  Magnaporthe grisea  comprising a gene coding for a protein (hereafter referred to as gene 763) whereof the presence and integrity are indispensable for pathogenesis of said fungus with respect to rice and barley. The invention also concerns the promoter of said gene, the gene coding for protein 763, protein 763 and uses thereof for identifying potential biological targets for novel fungicide molecules and for isolating genes coding for proteins controlling biochemical functions essential to the pathogenesis of the fungus  Magnaporthe grisea  with respect to rice and barley. The invention further concerns compounds inhibiting pathogenesis of fungi related to the expression of gene 763.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional of prior U.S. patent applicationSer. No. 10/240,363, which was filed on Apr. 14, 2003, now U.S. Pat. No.7,070,981 which is a 371 of PCT/FR01/00907 filed Mar. 26, 2001.

The present invention relates to a novel gene 763 which is essential tofungal pathogenesis. The invention relates to polynucleotides 763, topolypeptides 763, to host organisms expressing a polypeptide 763 and touses thereof for identifying novel antifungal molecules.

The principle of using genes of pathogenic fungi, entirely or in part,in tests for identifying novel molecules active against these fungi isin itself known (in Antifungal Agents: Discovery and Mode of Action, G.K. Dixon, L. G. Coppong and D. W. Hollomon eds, BIOS ScientificPublisher Ltd, Oxford UK). With this aim, knowledge of the genome of agiven pathogenic fungus constitutes an important step for theimplementation of such tests. However, the simple knowledge of a givengene is not sufficient to attain this objective, it also being necessaryfor the gene chosen as a target for potential fungicidal molecules to beessential to the life of the fungus, inhibition thereof by thefungicidal molecule leading to death of the fungus, or essential to thepathogenesis of the fungus, inhibition thereof not being lethal for thefungus but simply inhibiting its pathogenic capacity. This secondcategory of potential target genes for fungicidal molecules isparticularly important for the development of a new generation offungicidal products more favorable to the environment, whichspecifically attack only the pathogenic capacity of pathogenic fungi.

The present invention relates to the identification and cloning of anovel gene 763 which is essential for fungal pathogenesis. A mutant 763of Magnaporthe grisea, in which the gene is inactivated, exhibits apathogenesis reduced by 95%. Gene 763 encodes a transcription factorcomprising a motif of the bZIP type, composed of a dominant basicsequence-specific DNA binding motif followed by another termed “leucinezipper motif”, required for dimerization of the protein. This motif ischaracteristic of a vast family of proteins which regulate geneexpression. The expression of gene 763 is detected during the earlystages of plant infection. The invention also relates to the use of gene763 for identifying novel antifungal molecules and the use of gene 763for identifying other genes involved in fungal pathogenesis, theexpression of which is regulated by 763.

DESCRIPTION OF THE SEQUENCE LISTING

-   SEQ ID No. 1: Gene 763 of Magnaporthe grisea-   SEQ ID No. 2: eDNA of gene 763 of Magnaporthe grisea-   SEQ ID No. 3: Polypeptide 763 of Magnaporthe grisea-   SEQ ID No. 4: cDNA of gene 763 of Neurospora crassa-   SEQ ID No. 5: Polypeptide 763 of Neurospora crassa-   SEQ ID No. 6: partial sequence of protein 763 of Magnaporthe grisea    (SEQ ID No. 3)-   SEQ ID No. 7: partial sequence of transcription factor YAP-1-   SEQ ID No. 8: partial sequence of transcription factor GCN4-   SEQ ID No. 9: partial sequence of transcription factor CPC-1

DESCRIPTION OF THE INVENTION

Polynucleotides

The present invention relates to the polynucleotides comprising a fungalgene 763. Gene 763 can be isolated from phytopathogenic fungi such as,for example, Botrytis cinerea, Mycosphaerella graminicola, Stagnosporanodorum, Blumeria graminis, Colleotrichum lindemuthianum, Pucciniagraminis, Leptosphaeria maculans, Fusarium oxysporum, Fusariumgraminearum and Venturia inaequalis. Advantageously, gene 763 isisolated from phytopathogenic fungi of the genus Magnaporthe.Preferably, the polynucleotides of the invention comprise a gene 763 ofMagnaporthe grisea (SEQ ID NO: 1). Preferentially, the polynucleotidesof the present invention comprise the coding sequence of a gene 763 ofMagnaporthe grisea (SEQ ID NO: 2).

The term “polynucleotides 763” denotes all of the polynucleotides of thepresent invention, preferably the polynucleotides of the genomicsequence of 763, the polynucleotides of the cDNA sequence of 763, andalso the polynucleotides encoding the polypeptides 763 of the presentinvention. The term “polynucleotides 763” also denotes recombinantpolynucleotides comprising said polynucleotides.

According to the present invention, the term “polynucleotide” isintended to mean a single-stranded nucleotide chain, or the chaincomplementary thereto, or a double-stranded nucleotide chain, which maybe of the DNA or RNA type. Preferably, the polynucleotides of theinvention are of the DNA type, in particular double-stranded DNA. Theterm “polynucleotide” also denotes modified polynucleotides andoligonucleotides.

The polynucleotides of the present invention are isolated or purifiedfrom their natural environment. Preferably, the polynucleotides of thepresent invention can be prepared using the conventional molecularbiology techniques as described by Sambrook et al. (Molecular Cloning: ALaboratory Manual, 1989) or by chemical synthesis.

The invention relates to polynucleotides comprising the genomic sequenceof gene 763 of Magnaporthe grisea SEQ ID No. 1. This genomic sequencecomprises 4 exons (positions 723-770, 925-1194, 1273-1554 and 1663-1778of SEQ ID No. 1), 3 introns (positions 771-924, 1195-1272 and 1555-1662of SEQ ID No. 1), and 5′ and 340 regulatory sequences.

In a preferred embodiment of the invention, the polynucleotides of thegenomic sequence of 763 comprise a polynucleotide chosen from thefollowing polynucleotides:

-   a) the polynucleotide of SEQ ID No. 1,-   b) a polynucleotide comprising at least one exon of SEQ ID No. 1;-   c) a polynucleotide comprising a combination of exons of SEQ ID No.    1.

The present invention also relates to a polynucleotide comprising a 5′or 3′ regulatory sequence of gene 763 of Magnaporthe grisea. In a firstembodiment, the invention relates to a polynucleotide comprising thepromoter of gene 763 of Magnaporthe grisea, the sequence of which isincluded between position 1 and position 705 of SEQ ID No. 1. In anotherembodiment, the invention relates to a polynucleotide comprising abiologically active fragment of the promoter gene 763 of Magnaporthegrisea, the sequence of which is included between position 1 andposition 705 of SEQ ID No. 1.

The expression “biologically active fragment” is above intended to meana polynucleotide having promoter activity, and more particularlypromoter activity in fungi. The techniques which make it possible toevaluate the promoter activity of a polynucleotide are well known tothose skilled in the art. These techniques conventionally involve theuse of an expression vector comprising, in the direction oftranscription, the polynucleotide to be tested and a reporter gene (seeSambrook et al., Molecular Cloning: A Laboratory Manual, 1989).

The invention also relates to polynucleotides comprising the cDNA of 763of Magnaporthe grisea of SEQ ID No. 2. The cDNA gene 763 of Magnaporthegrisea comprises the coding sequence of gene 763 and also a 5′ UTRregulatory sequence and a 3′ UTR regulatory sequence. The invention moreparticularly relates to polynucleotides comprising the coding sequenceof gene 763 of Magnaporthe grisea, the sequence of which is includedbetween position 17 and position 733 of SEQ ID No. 2.

The invention also extends to the polynucleotides comprising apolynucleotide chosen from the following polynucleotides:

-   a) the polynucleotide of SEQ ID No. 1;-   b) the polynucleotide of SEQ ID No. 2;-   c) the polynucleotide of SEQ ID No. 4;-   d) a polynucleotide homologous to a polynucleotide as defined in a)    or b) or c);-   e) a polynucleotide capable of selectively hybridizing to a    polynucleotide as defined in a) or b) or c).

According to the invention, the term “homologous” is intended to mean apolynucleotide having one or more sequence modifications compared to thereference sequence. These modifications may be deletions, additions orsubstitutions of one or more nucleotides of the reference sequence.Advantageously, the percentage homology will be at least 70%, 75%, 80%,85%, 90%, 95% and preferably at least 98%, and more preferentially atleast 99%, relative to the reference sequence. The methods for measuringand identifying homologies between nucleic acid sequences are well knownto those skilled in the art. The PILEUP or BLAST programs (in particularAltschul et al., J. Mol. Evol. 36:290-300, 1993; Altschul et al., J.Mol. Biol. 215:403-10, 1990; Altschul et al., NAR 25:3389-3402, 1997)may, for example, be used. Preferably, the default parameters will beused. The invention therefore relates to polynucleotides comprisingpolynucleotides exhibiting at least 70%, 75%, 80%, 85%, 90%, 95%, 98%and preferably at least 98%, and more preferentially at least 99%,homology with the polynucleotides 763, the polynucleotides of SEQ IDNos. 1-2 or the polynucleotides of SEQ ID No. 4. Preferably, theinvention relates to a polynucleotide comprising a polynucleotide of atleast 50, 100, 200, 300, 400, 500, 1000 nucleotides exhibiting at least70%, 75%, 80%, 85%, 90%, 95%, 98% and preferably at least 98%, and morepreferentially at least 99%, homology with the polynucleotides 763, thepolynucleotides of SEQ ID Nos. 1-2 or the polynucleotides of SEQ ID No.4. Preferably, the polynucleotides homologous to a referencepolynucleotide conserve the function of the reference sequence.

According to the invention, the expression “sequence capable ofselectively hybridizing” is intended to mean the sequences whichhybridize with the reference sequence at a level significantly greaterthan the background noise. The level of the signal generated by theinteraction between the sequence capable of selectively hybridizing andthe reference sequences is generally 10 times, preferably 100 times moreintense than that of the interaction of the other DNA sequencesgenerating the background noise. Stringent hybridization conditionswhich allow selective hybridization are well known to those skilled inthe art. In general, the hybridization and washing temperature is atleast 5° C. below the Tm of the reference sequence at a given pH and fora given ionic strength. Typically, the hybridization temperature is atleast 30% for a polynucleotide of 15 to 50 nucleotides and at least 60°C. for a polynucleotide of more than 50 nucleotides. By way of example,the hybridization is carried out in the following buffer: 6×SSC, 50 mMTris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% ficoll, 0.02% BSA, and500 μg/ml denatured salmon sperm DNA. The washes are, for example,performed successively at low stringency in a 2×SSC, 0.1% SDS buffer, atmedium stringency in a 0.5×SSC, 01% SDS buffer and at high stringency ina 0.1×SSC, 0.1% SDS buffer. The hybridization may, of course, be carriedout according to other usual methods well known to those skilled in theart (see in particular Sambrook et al., Molecular Cloning: A LaboratoryManual, 1989). The invention therefore relates to polynucleotidescomprising a polynucleotide capable of selectively hybridizing with thepolynucleotide of SEQ ID Nos. 1-2 or the polynucleotide of SEQ ID No. 4.Preferably, the invention relates to a polynucleotide comprising apolynucleotide of at least 50, 100, 200, 300, 400, 500, 1000nucleotides, capable of selectively hybridizing with the polynucleotideof SEQ ID Nos. 1-2 or the polynucleotide of SEQ ID No. 4. Preferably,the polynucleotides which selectively hybridize to a referencepolynucleotide conserve the function of the reference sequence.

Preferably, the polynucleotides of the present invention conserve thefunction of gene 763 of Magnaporthe grisea (SEQ ID NO: 1) and encode atranscription factor which is essential to the pathogenesis of thefungus, and which is expressed at the beginning of the infectious state.

Preferentially, the polynucleotides of the present invention complementa mutant 763 of Magnaporthe grisea and restore its pathogenicity forrice and barley. A mutant 763 according to the invention is a mutant ofMagnaporthe grisea in which the gene 763 of SEQ ID No. 1 is inactivatedusing techniques well known to those skilled in the art.

The present invention also relates to allelic variants or homologues ofgene 763 of Magnaporthe grisea (SEQ ID NO: 1).

The present invention also relates to the identification and cloning ofgenes homologous to gene 763 of Magnaporthe grisea (SEQ ID NO: 1) inother phytopathogenic fungi. Preferably, these homologous genes can beisolated or cloned from a phytopathogenic fungus chosen from Botrytiscinerea, Mycosphaerella graminicola, Stagnospora nodorum, Blumeriagraminis, Colleotrichum lindemuthianum, Puccinia graminis, Leptosphaeriamaculans, Fusarium oxysporum, Fusarium graminearum and Venturiainaequalis. A subject of the invention is thus the use of apolynucleotide or of a fragment of a polynucleotide of SEQ ID No. 1 andof SEQ ID No. 2 according to the invention, for identifying homologousgenes in other phytopathogenic fungi. The techniques for cloninghomologous genes 763 in other phytopathogenic fungi are well known tothose skilled in the art. The cloning is carried out, for example, byscreening cDNA libraries or genomic DNA libraries with a polynucleotideor a fragment of a polynucleotide of SEQ ID No. 1 and of SEQ ID No. 2.These libraries can also be screened by PCR using specific or degenerateoligonucleotides derived from SEQ ID No. 1 or from SEQ ID No. 2. Thetechniques for constructing and screening these libraries are well knownto those skilled in the art (see in particular Sambrook et al.,Molecular Cloning: A Laboratory Manual, 1989). Phytopathogenic fungusgenes 763 may also be identified in the databases by nucleotide orprotein BLAST using SEQ ID Nos. 1-3.

Preferably, the cloned genes conserve the function of gene 763 ofMagnaporthe grisea (SEQ ID NO: 1 and encode a transcription factor whichis essential to the pathogenesis of the fungus, and which is expressedat the beginning of the infectious stage. The sequences of the clonedgenes can be analyzed according to known methods in order to establishthat they encode a fungal transcription factor and in particular inorder to establish that they encode a polypeptide comprising a motif ofthe bZIP type, composed of a dominant basic sequence-specific DNAbinding motif followed by another termed “leucine zipper motif”,required for dimerization of the protein. Moreover, the techniques forestablishing that a known gene is essential to the pathogenesis of afungus are known to those skilled in the art. For example, the genestudied is inactivated in the fungus using conventional molecularbiology techniques; mention will in particular be made of replacement ofthe gene with a marker gene by homologous recombination. The decrease inpathogenesis of the fungus comprising the inactivated gene is analyzedusing phenotypic tests. Preferably, the inactivation of the homologousgene causes a decrease in pathogenesis of at least 95%. The techniquesfor analyzing the expression of a gene in the various developmentalstages of the fungus, and more particularly at the beginning of aninfection, are also well known to those skilled in the art. Typically,total RNAs or mRNAs (poly A+) are prepared from the variousdevelopmental stages of the fungus. These RNAs are then analyzed byRT-PCR or by Northern Blotting in order to determine the level ofexpression of the gene. Other techniques well known to those skilled inthe art may be used in order to establish that the polynucleotides ofthe invention conserve the function of gene 763 of Magnaporthe grisea(SEQ ID NO: 1). Mention will be made in particular of complementation ofmutants 763 followed by tests for restoration of the pathogenesis of thefungus.

A blast search in databases made it possible to identify a homologue ofgene 763 of Magnaporthe grisea in Neurospora crassa. This novel gene wasidentified by blasting non-annotated genomic sequences. The cDNA of thisNeurospora gene 763 corresponds to SEQ ID No. 4 and the Neurosporapolypeptide 763 corresponds to SEQ ID No. 5.

A subject of the invention is also polynucleotides comprising apolynucleotide encoding a polypeptide chosen from the followingpolypeptides:

-   a) the polypeptide of SEQ ID No. 3;-   b) the polypeptide of SEQ ID No. 5;-   c) a polypeptide homologous to a polypeptide as defined in a) or b);-   d) a biologically active fragment of a polypeptide as defined in a)    or b).    Polypeptides

The present invention also relates to polypeptides 763 of aphytopathogenic fungus, and more particularly of Magnaporthe grisea. Theterm “polypeptides 763” denotes all the polypeptides of the presentinvention and also the polypeptides encoded by the polynucleotides ofthe present invention. The term “polypeptides 763” also denotes fusionproteins, recombinant proteins or chimeric proteins comprising thesepolypeptides. In the present description, the term “polypeptide” alsodenotes proteins and peptides, and also modified polypeptides.

The polypeptides of the invention are isolated or purified from theirnatural environment. The polypeptides may be prepared by variousmethods. These methods are in particular purification from naturalsources, such as cells naturally expressing these polypeptides,production of recombinant polypeptides by suitable host cells andsubsequent purification thereof, production by chemical synthesis or,finally, a combination of these various approaches. These variousmethods of production are well known to those skilled in the art. Thus,the polypeptides 763 of the present invention may be isolated from thefungi expressing polypeptides 763. Preferably, the polypeptides 763 ofthe present invention are isolated from recombinant host organismsexpressing a heterologous polypeptide 763. These organisms arepreferably chosen from bacteria, yeasts, fungi, animal cells or insectcells.

A subject of the present invention is a polypeptide comprising apolypeptide 763 of Magnaporthe grisea of SEQ ID No. 3. The inventionalso relates to polypeptides comprising a biologically active fragmentor a homologue of the polypeptide 763 of SEQ ID No. 3.

In another embodiment, a subject of the present invention is apolypeptide comprising a polypeptide 763 of Neurospora crassa of SEQ IDNo. 5. The invention also relates to polypeptides comprising abiologically active fragment or a homologue of the polypeptide 763 ofSEQ ID No. 5.

The term “fragment” of a polypeptide denotes a polypeptide comprisingpart but not all of the polypeptide from which it is derived. Theinvention relates to a polypeptide comprising a fragment of at least 10,15, 20, 25, 30, 35, 40, 50, 100, 200 amino acids of a polypeptide of SEQID No. 3.

The term “biologically active fragment” denotes a fragment of apolypeptide which conserves the function of the polypeptide from whichit is derived. The biologically active fragments of the polypeptide ofSEQ ID No. 3 thus conserve the function of the polypeptide 763 ofMagnaporthe grisea. These biologically active fragments therefore haveactivity of a transcription factor which is functional in fungi.Preferentially, this activity is essential to the pathogenesis of thefungus.

The term “homologue” denotes a polypeptide which may have a deletion, anaddition or a substitution of at least one amino acid. A subject of theinvention is a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 98%and preferentially at least 99% of amino acids identical with apolypeptide of SEQ ID No. 3 or of SEQ ID No. 5. The methods formeasuring and identifying homologies between polypeptides or proteinsare known to those skilled in the art. The UWGCG package and theBESTFITT program may, for example, be used to calculate the homologies(Devereux et al., Nucleic Acid Res. 12, 387-395, 1984). The defaultparameters are preferably used.

Preferably, these homologous polypeptides conserve the same biologicalactivity as the polypeptide 763 of Magnaporthe grisea of SEQ ID No. 3.Preferentially, these polypeptides therefore have a fungal transcriptionfactor activity. Preferentially, this activity is essential to thepathogenesis of the fungus. In a preferred embodiment, these homologouspolypeptides may be isolated from phytopathogenic fungi. Preferably,these polypeptides are expressed in phytopathogenic fungi at thebeginning of plant infection.

A subject of the invention is also a fusion polypeptide comprising apolypeptide 763 as described above fused to a reporter polypeptide. Thereporter polypeptide allows rapid detection of the expression of apolypeptide 763 in a fungus or in another host organism. Among thepolypeptides which may thus be fused with a polypeptide 763, mentionwill be made in particular of GFP (green fluorescent protein) and theGUS (β-glucuronidase) protein. These fusion proteins and theirconstructs are well known to those skilled in the art.

Expression Cassettes, Vectors and Host Organisms

Gene 763 can be expressed in various host organisms, such as bacteria,yeasts, fungi, animal cells or insect cells. Gene 763 can be expressedin a host organism under the control of the promoter 763 of the presentinvention or under the control of a heterologous promoter.

Expression Cassettes

According to an embodiment of the invention, a polynucleotide encoding apolypeptide 763 is inserted into an expression cassette using cloningtechniques well known to those skilled in the art. This expressioncassette comprises the elements required for the transcription andtranslation of the sequences encoding the polypeptide 763.Advantageously, this expression cassette comprises both elements formaking a host cell produce a polypeptide 763 and elements required forregulating this expression. In a first embodiment, the expressioncassettes according to the invention comprise, in the direction oftranscription, a promoter which is functional in a host organism, gene763 or the sequence encoding gene 763, and a sequence which is aterminator sequence in said host organism. Preferentially, theexpression cassette comprises, in the direction of transcription, apromoter which is functional in a host organism, a polynucleotide chosenfrom the following polynucleotides:

-   a) a polynucleotide encoding the polypeptide 763 of SEQ ID No. 3 or    encoding a biologically active fragment of the polypeptide 763 of    SEQ ID No. 3;-   b) a polynucleotide, the sequence of which is included between    position 17 and position 733 of SEQ ID No. 2;-   c) a polynucleotide of SEQ ID No. 1;-   d) a polynucleotide of SEQ ID No. 2;-   e) a polynucleotide encoding the polypeptide 763 of SEQ ID No. 5 or    encoding a biologically active fragment of the polypeptide 763 of    SEQ ID No. 5;-   f) a polynucleotide of SEQ ID No. 4;-   g) a polynucleotide homologous to a polynucleotide as defined in b),    c), d) or f);-   h) a polynucleotide capable of specifically hybridizing to a    polynucleotide as defined in b), c), d) or f);    and a sequence which is a terminator sequence in said host organism.

Any type of promoter sequence may be used in the expression cassettesaccording to the invention. The choice of promoter will in particulardepend on the host organism chosen for expressing the gene of interest.Some promoters allow constitutive expression whereas other promotersare, on the contrary, inducible. Among the promoters which arefunctional in fungi, mention will be made in particular of that ofAspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (Robertset al., Current Genet. 15:177-180, 1989). Among the promoters which arefunctional in bacteria, mention will be made in particular of the T7bacteriophage RNA polymerase (Studier et al., Methods in enzymology185:60-89, 1990). Among the promoters which are functional in yeasts,mention will be made in particular of that of the Gall gene (Elledge etal., Proc. Nat. Acad. Sciences, USA. 88:1731-1735, 1991) or the GAL4 andADH promoters of S. cerevisiae. Among the promoters which are functionalin insect cells, mention will be made in particular of the polyhedrinpromoter of the baculovirus AcMNPV (Weyer et al., J. Gene. Virol.72:2967-2974, 1991). Among the promoters which are functional in animalcells, mention will be made of the metallothionein promoter and viraland adenoviral promoters. All these promoters are described in theliterature and are well known to those skilled in the art.

The promoter 763 may be used to express a heterologous gene in a hostorganism and in particular in fungi. A subject of the invention istherefore also expression cassettes comprising the promoter of a gene763, functionally associated with a sequence encoding a heterologousprotein, allowing expression of said protein in fungi. Preferably, theexpression cassette according to the invention comprises, in thedirection of transcription, a polynucleotide, the sequence of which isincluded between position 1 and position 705 of SEQ ID No. 1, or abiologically active fragment of the polynucleotide, the sequence ofwhich is included between position 1 and position 705 of SEQ ID No. 1,the sequence encoding a heterologous polypeptide and terminator sequencewhich is functional in fungi. Any gene of interest may be expressed in ahost organism under the control of a promoter 763. Preferably, thepromoter 763 is used for expressing a heterologous gene in fungi. Theactivity of the promoter 763 under various conditions may be evaluatedusing a reporter gene such as the GUS (β-glucuronidase), GFP (greenfluorescent protein), LUC (luciferase), CAT (chloramphenicoltransferase) or β-galactosidase (lacZ) reporter gene.

In a preferred embodiment of the invention, the promoter 763 isfunctionally associated with the coding sequence of a marker gene.Expression of the marker gene allows the transformed organisms to beselected by virtue of their resistance to antibiotics or to herbicidesfor example. Mention will in particular be made of the coding sequencesfor a gene for tolerance to an antibiotic or a herbicide, such as thegenes for resistance to hygromycin (hph: Punt et al., 1987), tobleomycin (ble: Drocourt, 1990) or to the herbicide bialaphos (Bar: Palland Brunelli, 1993).

The expression cassettes according to the present invention may alsoinclude any other sequence required for expressing gene 763 or theheterologous gene, such as, for example, regulatory elements or signalsequences for addressing the polypeptide 763. Any regulatory sequencemaking it possible to increase the level of expression of the codingsequence inserted into said expression cassette may in particular beused. According to the invention, it is in particular possible to use,in combination with the promoter regulatory sequence, other regulatorysequences, which are located between the promoter and the codingsequence, such as transcription activators (enhancers). As a signal formembrane addressing in the host organisms, mention will in particular bemade of that of protein A in bacteria (Nilsson et al., Methods inEnzymology 198:3, 1991).

A large variety of terminator sequences can be used in the expressioncassettes according to the invention, these sequences allowingtermination of transcription and polyadenylation of the mRNA. Anyterminator sequence which is functional in the host organism selectedmay be used.

A subject of the present invention is also a polynucleotide comprisingan expression cassette according to the invention; advantageously, theexpression cassettes according to the present invention are insertedinto a vector.

Vectors

The present invention therefore also relates to replication orexpression vectors for transforming a host organism, comprising at leastone polynucleotide 763 or an expression cassette according to thepresent invention. This vector may in particular consist of a plasmid, acosmid, a bacteriophage or a virus, into which a polynucleotide 763 oran expression cassette according to the invention is inserted. Thetechniques for constructing these vectors and for inserting apolynucleotide of the invention into these vectors are well known tothose skilled in the art. In general, any vector capable of maintainingitself, of self-replicating or of propagating in a host cell, and inparticular in order to induce the expression of a polynucleotide or of apolypeptide, may be used. Advantageously, the vectors according to theinvention comprise at least one origin of replication in order for themto replicate in a host organism. Preferably, the vectors of theinvention also comprise at least one selectable marker, such as a genefor resistance to an antibiotic. Mention will in particular be made ofvectors such as pBluescript (Stratagene, La Jolla, Calif.), pTrcHis(Invitrogen, La Jolla, Calif.) and baculovirus-derived expressionvectors, such as those derived from the Autographica californicapolyhedrovirus (AcMNPV). A preferred system combining a baculovirus andan insect cell is the pV111392 baculovirus/Sf21 cell system (Invitrogen,La Jolla, Calif.). For expression in animal cells, adenovirus-derivedvectors are in particular used. Those skilled in the art will choose thesuitable vectors in particular as a function of the host organism to betransformed and as a function of the transformation technique used. Themethods for transforming host organisms are well known to those skilledin the art (Inoue et al., Gene 96:23-28, 1990; Fincham, MicrobiologicalReviews 53:148-170, 1989).

The vectors of the present invention are in particular used to transforma host organism for the purpose of replication of the vector and/orexpression of a polypeptide 763 in said host organism. The inventionrelates to a method for preparing a polypeptide M763, comprising thefollowing steps:

-   -   a host organism is transformed with an expression vector        comprising an expression cassette according to the invention,    -   the polypeptides M763 produced by the host organism are        isolated.

The recombinant polypeptides 763 produced by a host organism transformedwith a polynucleotide can be purified or isolated according to methodsknown to those skilled in the art. The polypeptides M763 can beexpressed in a host organism in the form of fusion proteins. Mentionwill in particular be made of the vectors pGEX for expressing fusionproteins comprising glutathione S-transferase (GST). These fusionproteins are easily purified by adsorption on glutathione-agarose beads.The GST group can then be removed by digestion with protease Xa. Othersystems for expressing and purifying fusion proteins are known to thoseskilled in the art.

Host Organisms

A subject of the present invention is also a method for transforming ahost organism by integrating into said host organism at least onepolynucleotide 763 or an expression cassette or a vector according tothe invention. The polynucleotide may be integrated into the genome ofthe host organism or may replicate stably in the host organism. Themethods for transforming host organisms are well known to those skilledin the art and widely described in the literature (Inoue et al., Gene96:23-28, 1990; Fincham, Microbiological Reviews 53:148-170, 1989).

The present invention also relates to a host organism transformed with apolynucleotide 763, an expression cassette or a vector according to theinvention. According to the invention, the term “host organism” is inparticular intended to mean any lower or higher, unicellular orpluricellular organism, in particular chosen from bacteria, yeasts,fungi, animal cells and insect cells. Advantageously, the bacteria arechosen from Escherichia coli and Bacillus subtilis, the yeasts arechosen from Pichia pastoris and Saccharomyces cerevisae, the insectcells are chosen from Spodoptera frugiperda and Drosophila melanogaster,and the animal cells are chosen from CHO, HeLa and COS cells.

The techniques for constructing vectors, for transforming host organismsand for expressing heterologous proteins in these organisms are widelydescribed in the literature (Ausubel F. M. et al., “Current Protocols inMolecular Biology” Volumes 1 and 2, Greene Publishing Associates andWiley-Interscience, 1989; T. Maniatis, E. F. Fritsch, J. Sambrook,Molecular Cloning A Laboratory Handbook, 1982).

The present invention also relates to the use of polynucleotides 763 andof polypeptides 763 for identifying genes involved in fungalpathogenesis and for identifying novel fungicidal molecules whichinhibit fungal pathogenesis.

Inhibition of Fungal Pathogenesis

Fungi in which gene 763 is inactivated or inhibited exhibit apathogenesis which is reduced by 95%. The invention relates to methodsfor inhibiting fungal pathogenesis by inactivating or inhibiting theexpression of gene 763. Preferably, the fungi are chosen from Botrytiscinerea, Mycosphaerella graminicola, Stagnospora nodorum, Blumeriagraminis, Colleotrichum lindemuthianum, Puccinia graminis, Leptosphaeriamaculans, Fusarium oxysporum, Fusarium graminearum and Venturiainaequalis.

Preferably, the invention relates to methods for inhibiting thepathogenesis of a fungus, said methods comprising inhibiting theexpression of a polynucleotide 763 according to the invention in saidfungus, or inhibiting the expression of a polypeptide 1763 according tothe invention in said fungus or inhibiting the biological activity of apolypeptide 763 according to the invention in said fungus. Preferably,this inhibition affects specifically the expression of gene 763 and thebiological activity of the polypeptide 763. The invention does nottherefore relate to the methods comprising the general inhibition ofgene expression in the fungus. It will be understood that the inhibitionof the expression of gene 763 may, however, lead to the inhibition ofother genes.

Various methods well known to those skilled in the art may be used toinhibit fungal pathogenesis by inhibiting the expression of gene 763 inthese fungi. In one embodiment of the invention, gene 763 is inactivatedby insertional mutagenesis or by homologous recombination (genereplacement or “knock out” techniques). In another embodiment of theinvention, the expression of a polypeptide 763 is inhibited byexpressing an antisense polynucleotide of gene 763 in the fungi. In athird embodiment of the invention, the expression of gene 763 isinhibited by an inhibiting compound.

The level of expression of a polynucleotide 763 or of a polypeptide 763in the fungi can be measured according to techniques described in theliterature. Mention will in particular be made of Northern blotting, PCRand DNA arrays (DNA chips) for the polynucleotides and Western blottingfor the polypeptides. These techniques are well known to those skilledin the art.

Identification of Novel Fungicidal Molecules which Inhibit FungalPathogenesis

Inactivation of gene 763 in Magnaporthe grisea, a pathogenic fungus ofrice, leads to a 95% decrease in the pathogenesis of this fungus.Moreover, it has been possible to identify homologous genes in otherfungi. Consequently, compounds which inhibit the expression of gene 763or the activity of the polypeptide 763 in fungi can be used to inhibitfungal pathogenesis.

The invention therefore relates to methods for identifying compoundswhich inhibit fungal pathogenesis, comprising a step of identifying acompound which specifically inhibits the expression of a polynucleotide763 in said fungus, or a step of identifying a compound which inhibitsthe expression of a polypeptide 763 in said fungus or a step ofidentifying a compound which inhibits the biological activity of apolypeptide 763 in said fungus. Preferably, the fungi are chosen fromBotrytis cinerea, Mycosphaerella graminicola, Stagnospora nodorum,Blumeria graminis, Colleotrichum lindemuthianum, Puccinia graminis,Leptosphaeria maculans, Fusarium oxysporum, Fusarium graminearum andVenturia inaequalis.

The polynucleotides 763, the polypeptides 763, the vectors and the hostorganisms of the present invention may thus be used in various screeningassays in order to identify novel antifungal compounds.

Identification of Inhibitors which Bind to the Protein 763

Molecules which directly inhibit the activity of the polypeptide 763might inhibit the pathogenesis of the fungus and lead to the developmentof novel fungicides.

The invention therefore relates to a method for identifying compoundswhich inhibit fungal pathogenesis, comprising the following steps:

-   -   bringing said compound into contact with a polypeptide 763, and    -   detecting the binding of said compound to said polypeptide; and        preferentially, the method also comprises a step in which it is        determined whether said compound inhibits fungal pathogenesis.

Any method for preparing a polypeptide 763 and for purifying it or forisolating it may be used in the methods of the present invention.

Preferably, the polypeptide 763 is expressed in a heterologousexpression system (for example bacterium, yeast, animal cell or insectcell) by means of a polynucleotide 763 according to the invention; thesimplified purification of the polypeptide 763 then makes it possible toidentify novel molecules which bind to the protein 763. Said moleculesare identified using methods well known to those skilled in the art, inparticular methods of physical detection of the binding of the compoundstested to the protein 763 (BIACORE system; Karlson & al., J. ofBiomolecular Interaction Analysis, Special Issue Drug Discovery: 18-22).

Identification of Inhibitors of Gene 763 Expression Regulators

Molecules which inhibit the expression of gene 763 may also inhibit thepathogenesis of the fungus and lead to the development of novelfungicides. In the present invention, the expression “inhibition of theexpression of gene 763” denotes the inhibition of the expression of apolynucleotide 763 and also the inhibition of the expression of apolypeptide 763 in host organisms, and preferentially in phytopathogenicfungi.

A subject of the invention is also a method for identifying compoundswhich inhibit fungal pathogenesis, comprising the following steps:

-   -   bringing said compound into contact with a host organism        transformed with a polynucleotide or a vector according to the        invention such that this host organism expresses a reporter gene        under the control of the promoter of gene 763; and    -   detecting the inhibition of the expression of said reporter        gene.

Preferentially, the method also comprises a step in which it isdetermined whether said compound inhibits fungal pathogenesis.

The use of a polynucleotide according to the invention, comprising thepromoter 763 associated with the coding sequence of a reporter gene (GUSor GFP for example) makes it possible to measure the promoter activityof the promoter 763 in a fungal cell or in a host cell. This methodmakes it possible to identify compounds which inhibit the activity ofthe promoter 763 and therefore the expression of gene 763 at thetranscriptional level. A recombined strain comprising the above gene isthus used to identify molecules which inhibit the expression of gene763, which manifests itself by inhibition of the expression of thereporter protein of the recombined strain under conditions forexpression of gene 763. This type of assay is well known to thoseskilled in the art and described in the literature, in particularAxiotis et al. (1995. pp. 1-7 in Antifungal Agents: Discovery and Modeof Action. G. K. Dixon, L. G. Coppong and D. W. Hollomon, eds, BIOSScientific Publisher Ltd. Oxford, UK).

In another embodiment, the invention relates to a method for identifyingcompounds which inhibit fungal pathogenesis, comprising the followingsteps:

-   -   bringing said compound into contact with a host organism        transformed with a polynucleotide according to the invention or        a vector according to the invention, said host organism        expressing a polypeptide 763; and    -   detecting the inhibition of the expression of said polypeptide        763.

Preferably, the polypeptide 763 is a fusion polypeptide comprising areporter polypeptide such as GUS of GFP, the expression of which iseasily measured. Preferentially, the method also comprises a step inwhich it is determined whether said compound inhibits fungalpathogenesis. This method makes it possible to identify compounds whichinhibit the expression of gene 763 at the transcriptional level or atthe translational level. A recombined strain expressing a polypeptide763, and preferably a polypeptide 763 fused to a reporter, is thus usedto identify molecules which inhibit the expression of gene 763, whichmanifests itself by inhibition of the expression of the polypeptide 763of the recombined strain under the conditions for expression of gene763.

The present invention therefore relates to a method for identifyingcompounds which inhibit fungal pathogenesis associated with expressionof gene 763, said method consisting in subjecting a compound, or amixture of compounds, to an assay suitable for identifying compoundswhich inhibit said fungal pathogenesis, and in selecting the compoundswhich react positively to said assay and, where appropriate, inisolating them and then in identifying them.

Preferentially, the suitable assay is an assay as defined above.

Preferably, a compound identified according to these methods is thentested for its antifungal properties and for its ability to inhibit thepathogenesis of the fungus for plants, according to methods known tothose skilled in the art. Preferentially, the compound is evaluatedusing phenotypic tests, such as pathogenesis assays on leaves or onwhole plants.

According to the invention, the term “compound” is intended to mean anychemical compound or mixture of chemical compounds, including peptidesand proteins.

According to the invention, the expression “mixture of compounds” isunderstood to mean at least two different compounds, such as, forexample, the (dia)stereoisomers of a molecule, mixtures of naturalorigin derived from the extraction of biological material (plants, planttissues, bacterial cultures, yeast or fungal cultures, insects, animaltissues, etc.) or reaction mixtures which are unpurified or totally orpartly purified, or else mixtures of products derived from combinatorialchemistry techniques.

Finally, the present invention relates to novel compounds which inhibitfungal pathogenesis associated with expression of gene 763, inparticular the compounds identified by the method according to theinvention and/or the compounds derived from the compounds identified bythe method according to the invention.

Preferentially, the compounds which inhibit fungal pathogenesisassociated with expression of gene 763 are not general enzymeinhibitors. Also preferentially, the compounds according to theinvention are not compounds already known to have fungicidal activityand/or activity on fungal pathogenesis.

A subject of the invention is also a method for treating plants againsta phytopathogenic fungus, characterized in that it comprises treatingsaid plants with a compound identified by a method according to theinvention.

The present invention also relates to a method for preparing a compoundwhich is an inhibitor of fungal pathogenesis, said method comprising thesteps of identifying a compound which inhibits fungal pathogenesisassociated with the expression of gene 763, by the identification methodaccording to the invention, and then preparing said identified compoundby the usual methods of chemical synthesis, of enzymatic synthesisand/or of extraction of biological material. The step of preparing thecompound may be preceded, where appropriate, by an “optimization” stepby which a compound derived from the compound identified by theidentification method according to the invention is identified, saidderived compound then being prepared by the usual methods.

The examples below make it possible to illustrate the invention without,however, seeking to limit the scope thereof.

All the methods or operations described below in these examples aregiven by way of examples and correspond to a choice, made from thevarious methods available for achieving the same result. This choice hasno bearing on the quality of the result and, consequently, any suitablemethod may be used by those skilled in the art in order to achieve thesame result. Most of the DNA fragment engineering methods are describedin “Current Protocols in Molecular Biology” Volumes 1 and 2, F. M.Ausubel et al., published by Greene Publishing Associates andWiley-Interscience (1989), or in Molecular Cloning, T. Maniatis, E. F.Fritsch and J. Sambrook (1982). The methods specific for fungi aredescribed in Sweigard et al. (Fungal Genetics Newsletter, 44:52-53,1997) for the fungal transformation vectors used, in Orbach (Gene150:159-162, 1994) for constructing a cosmid library, in Sweigard et al.(Fungal Genetics Newsletter, 37:4-5, 1990) for preparing fungal genomicDNAs, and in Agnan et al. (Fungal Genetics and Biology, 21:292-301,1997).

DESCRIPTION OF THE FIGURES

FIG. 1: Autoradiogram of hybridization, with a probe pAN7.1, of thetransfer onto nylon membranes of genomic DNA digestions of the mutant763. (E:EcoRI; A:ApaI; C:ClaI; K:KpnI).

FIG. 2: “Plasmid rescue” in the mutant 763. Genomic DNA of the mutant763 (in bold) with insertion site of the plasmid. The positions of theEcoRI and KpnI sites on the genomic DNA are arbitrary.

FIG. 3: Insertion locus of the plasmid pAN7.1 and BglII-XhoI restrictionfragment (6 kb) complementing the mutant m763. The position of thegenomic probe (0.4 kb) derived from PRK763 is indicated in bold. Thearrows indicate the position of the PCR primers for amplifying the pointof insertion of the plasmid into the wild-type strain. The point ofinsertion of the plasmid pAN7.1 is also indicated.

FIG. 4: Identification of a basic “leucine zipper” domain. Consensusobtained by alignment of the sequence of the protein P763 with those ofthe transcription factors YAP-1 and GCN4 of Saccharomyces cerevisiae andMEAB of Aspergillus nidulans. This domain comprises a basic domain (A)and a “leucine zipper” domain per se (B).

FIG. 5: Consensus obtained by alignment of the sequence of the protein763 of Magnaporthe grisea (SEQ ID NO: 6) with those of the transcriptionfactors YAP-1 (SEQ ID NO: 7) and GCN4 (SEQ ID NO: 8) of Saccharomycescerevisiae and CPC-1 (SEQ ID NO: 9) of Neurospora crassa.

FIGS. 6A and 6B: Autoradiograms of hybridization, with a probeconsisting of the cDNA of gene 763, of Southern membranes of theproducts of RT-PCR and nested-PCR amplification of the mRNA of this geneunder various conditions.

FIG. 7: Alignment of the protein 763 of Magnaporthe grisea (SEQ ID NO:3) and of the homologous protein of Neurospora crassa (SEQ ID NO: 5).

Alignment produced using the clustal-W program.

(*: identical amino acids).

EXAMPLES

The strategy employed to achieve the identification and characterizationof gene 763 essential to the pathogenesis of M. grisea comprised twomain points:

-   1) Inactivation of a gene essential to pathogenesis by random    insertion into its nucleotide sequence of a foreign DNA fragment    (insertional muta-genesis).-   2) Recovery and characterization of the fungal nucleotide sequence    thus modified, and then demonstration of its involvement in the    pathogenesis of the fungus with respect to rice and to barley.

The methodological steps to be successively surmounted are as follows:

-   1) Obtaining a collection of fungal isolates having randomly    integrated a foreign DNA fragment into their genome (transformants).    In this case, the foreign DNA is a plasmid comprising the hph gene    of Escherichia coli, which allowed them to be selected on the basis    of hygromycin resistance. It was introduced into the fungal genome    by protoplast transformation.-   2) Searching for transformants which are nonpathogenic with respect    to rice and to barley, among the collection (pathogenesis mutants).    The criterion selected for nonpathogenesis of a transformant was the    inability to cause foliar lesions subsequent to inoculation of    spores of this transformant into rice and barley plants.-   3) Genetically demonstrating the inactivation of a pathogenesis gene    by the plasmid in the mutants incapable of infecting rice and    barley. This involved establishing complete genetic linkage between    the hygromycin-resistance characteristic, which reflects the    presence of the plasmid in the genome of the mutant, and that of    nonpathogenesis, which reflects the inactivation of a gene essential    to the infectious capacity of the fungus. This degree of linkage was    evaluated by analysis of segregation of the hygromycin-resistance    and nonpathogenesis characteristics in the descendents of a cross    between the mutant studied and a wild-type strain pathogenic with    respect to rice and to barley and having a mating type compatible    with that of the mutant.-   4) Recovering the genomic region of the fungus at which the    insertion of the mutating plasmid occurred. The principle consisted    in isolating a DNA fragment of the mutant comprising both plasmid    and genomic sequences, detectable by virtue of a hybridization    experiment with a probe of plasmid origin. The genomic component    included in this fragment was then used to isolate the complete    wild-type genomic region according to the same principle.-   5) Demonstrating that the genomic region next to the point of    insertion of the plasmid contains the pathogenesis gene. If the    pathogenesis gene sought is in the genomic region next to the point    of insertion of the plasmid, the introduction thereof into the    genome of the mutant isolate, using a plasmid vector comprising    another selectable marker, should make it possible to restore    pathogenesis by complementation of the function made deficient by    insertion of the first plasmid. Proof of this is provided if the    spores of at least one transformant obtained through this experiment    are capable of causing as many foliar lesions as the wild-type    strain.-   6) Characterizing the genomic sequence of the fungus in the    proximity of the point of insertion of the plasmid. The product from    sequencing the genomic region next to the point of insertion of the    plasmid is analyzed with sequence processing programs, so as to    attempt to demonstrate therein a nucleotide sequence capable of    being translated into peptide sequence (open reading frame). This    search is carried out on the basis of searching for consensus    signals for initiation and termination of translation to protein.    Proof of the existence of an open reading frame (and therefore of a    gene) in this region was provided by cloning the corresponding    transcriptional unit, by screening a library of DNAs complementary    to messenger RNAs (cDNAs) with a probe produced from a fragment of    this region. The sequence of this cDNA makes it possible to    determine with precision the size and the primary sequence of the    corresponding protein, and also the position of possible introns in    the genomic sequence of the gene.

Example 1 Insertional Mutagenesis

Protoplast transformation with an integrative plasmid carrying aselectable marker was used as an insertional mutagenesis tool in orderto search for the pathogenesis genes of the rice-parasite ascomycetefungus Magnaporthe grisea. The conditions for culturing, for obtainingprotoplasts, for transformation and also for purifying and storingMagnaporthe grisea transformants are described by Silué et al. (Physiol.Mol. Plant Pathol., 53, 239-251, 1998). The transformation was carriedout with 1 μg of plasmid pAN7.1 (Punt et al., Gene 78: 147-156), 1987)and 10⁷ protoplasts of the M. grisea strain P1.2. This strain originatesfrom the collection of the phytopathology laboratory of the CIRAD[International Center for Cooperation in Agronomic Research forDevelopment] in Montpellier. The transformants were selected byincorporating hygromycin into the agar culture media, at theconcentrations of 240 ppm for the primary selection medium and of 120ppm for the secondary selection medium.

Example 2 Screening the Collection of Transformants and Identifying theNonpathogenic Mutant 763

A) Pathogenesis Assays on Leaves Under Survival Conditions

The pathogenesis assays were carried out on two varieties of rice,Maratelli and Sariceltick, and one variety of barley, Express. Maratelliare varieties which are very sensitive to blast disease and which do nothave genes for resistance to the strain P1.2. The barley varieties areextremely sensitive to blast disease. The rice was grown at 25° C.during the day and 15° C. at night with a hygrometry of greater than70%, the barley was grown under cold conditions (20-22° C.). Rice andbarley leaf fragments (2.5 cm) were removed from the median part of theyoungest leaf of plants about twenty days old. These fragments wereplaced in multicompartment dishes containing water with 1% agarsupplemented with 2 mg/l of kinetin, a medium which allows them tosurvive for 14 days. It is important to note that rice develops a strongphysiological resistance to blast disease during periods of great heat.This resistance may be attenuated by giving the plants nitrogen-basedfertilizer: two waterings with a solution of ammonium sulfate at 5 g/m²,one week apart. The second watering takes place 2 to 3 days beforeinoculation.

The conditions for sporulation and for preparing M. grisea sporeinoculum are described by Silué et al. (mentioned above). Theinoculation was performed using a wet cotton-wool bud soaked in asuspension of spores and passed over the leaf fragments under survivalconditions. The amount of spores deposited was estimated by depositing adrop of the suspension onto a glass slide. The symptoms were observedafter 4-7 days of incubation at 24° C., 100% hygrometry. Eachtransformant was tested on four rice leaf fragments of each variety andfour of barley during the first screening. The transformant 763 shows adecrease in pathogenesis quantified at 95% of the number of lesionscaused by the wild-type strain. The transformant 763 was inoculated asecond time, in order to confirm its phenotype, with a suspension ofspores having a concentration adjusted to 10⁵ spores per ml. The resultsare given in the table below.

TABLE 1 Penetration of the mutant 763 into barley leaves Inoculation ofbarley leaves with drops of 35 microliters containing spores (500000spores/ml) Exp. 1 48 h after inoculation, many surface appressoria, fewpenetrations, some infectious hyphae visible, 6 days, no visible lesion,brown coloration at the point of contact of the drop Exp. 2 48 h afterinoculation, many surface appressoria, penetration not observed 4 days,no visible lesion, brown coloration at the point of contact of the dropExp. 3 48 h after inoculation, many surface appressoria, fewpenetrations, infectious hyphae visible in the leaf, colonizationgreatly slowed compared to P12B) Pathogenesis Assays on Whole Plants

In order to confirm the phenotype of the nonpathogenic mutant 763detected by inoculation of leaves under survival conditions, thedepartment of phytopathology of the CIRAD at Montpellier performedinoculations of whole plants with the spores of this mutant. The tworice cultivars sensitive to the P1.2 strain, Maratelli and Sariceltickwere sown and cultured under glass. Three nitrogen applications wereperformed during the first three weeks of culturing (at 5, 10 and 20days after sowing). The inoculation by spraying a suspension of sporestakes place 10 to 15 days after giving nitrogen for the last time,depending on the degree of maturity of the plants. The sporeconcentration was determined by counting with a Thoma cell and adjustedto a value of 20 000 spores/ml. The suspensions of spores of the mutant763 and of the nontransformed strain P1.2 were sprayed onto thirtyplants, in a proportion of 1 ml of spore suspension per plant, with anaerograph. One leaf from each of these plants was collected for countingthe number of lesions, after they had developed (5 to 7 days). A 93%decrease in pathogenesis was also observed on whole plants (see tablebelow).

TABLE 2 Spraying a spore suspension onto whole plants (rice, varietySariceltick) Decrease compared P12 (wild-type strain) Mutant 763 to P12Exp. 1 Spores 42 lesions per leaf 7 lesions per leaf −85% 25000 sp/mllesion size: 3.5 mm² lesion size: 0.5 mm² −85% Exp. 2 Spores 30 lesionsper leaf 2 lesions per leaf −93% 100000 sp/ml

Example 3 Phenotypic Analysis of the Mutant 763

The mutant 763 is affected by a decrease in pathogenesis quantified at93% of the number of lesions caused by the wild-type strain, without itsability to sporulate being lessened. In addition, while the rare lesionsobserved were clearly visible and made up of a necrotic area surroundedby a brownish border (typical symptom of blast disease), they were allsmall in size and nonsporulating, contrary to those caused by thewild-type strain (−90% at the surface). An infection assay on injuredleaves shows that the progression of the hyphae of this mutant, inplanta, remains limited to the area of injury. Cytological analysis ofthe infection in this mutant shows that the mutant manages to penetratethrough the epidermal cell wall in barley, but it is then rapidlyblocked in its progression.

The physiology and morphology of the conidia and of the mycelium of thetransformant 763 are apparently normal. Its ability to differentiateappressoria on barley epidermis, as on artificial hydrophobic surfaces(PVC, Teflon, PET), is not different from that of the wild-type strain.

The growth of this mutant was also studied in the presence of salts anddrugs which interfere with assimilation of nitrogen compounds. Thisinvolved determining whether it exhibited the phenotype of loss ofmetabolic repression of nitrogen, a characteristic of the mutant meaB ofAspergillus nidulans (see later, molecular analysis; Polley and Caddick,1996). In this fungus, the metabolic repression of nitrogen results, inthe presence of ammonium or of L-glutamine, in inhibition of the genesrequired for acquiring and using other nitrogen sources. The mutationmeaB is characterized by its resistance to methylammonium (a toxicinducer of metabolic repression of nitrogen) but also by its resistanceto parafluorophenylalanine and by its hypersensitivity to nitrictoxicity. The mutant 763 does not have a phenotype different from thatof the wild-type strain under all the conditions tested. 763 also growsnormally on minimum medium (MM).

Example 4 Genetic and Molecular Analysis of the Mutant 763

20 ascospores at random and a tetrad derived from the cross M4×763 wereanalyzed. The results of this analysis appear in the table below andshow that hygromycin resistance cosegregates with loss of pathogeniccapacity: the mutated pathogenesis gene is tagged with the plasmidpAN7.1 in 763.

TABLE 3 Analysis of the descendents of the cross M4 × 763 Ascospore Hyg.Path. Parenteral tetrad 1 s + 2 s + 3 s + 4 s + 5 R − 6 R − 7 R − 8 N.DN.D. Ascospores at random 1 s + 2 N.D. N.D. 3 R − 4 R − 5 R − 6 R − 7 R− 8 S + 9 R − 10 R − 11 R − 12 s + 13 s + 14 s + 15 s + 16 R − 17 R − 18R − 19 s + 20 s +

The number of copies of the plasmid pAN7.1 present in the genome of thistransformant and the relative position of the point of integration weredetermined by hybridization with a plasmid probe (see FIG. 1). Threetypes of restriction enzyme were used depending on the number ofcleavages desired: EcoRI (2 cleavages); BamHI (1 cleavage); ApaI, ClaIand KpnI (no cleavage). The hybridization profiles of the restrictionfragments obtained show that this transformant comprises only one copyof the plasmid (3 EcoRI fragments, a single fragment for BamHI, ApaI,ClaI and KpnI). Moreover, the single BamHI hybridization fragment isgreater than 6.75 kb in size. This indicates that the copy of theplasmid integrated into the genome of this transformant does not possessat its ends the two BamHI sites expected subsequent to transformation inthe presence of this restriction enzyme. According to the analysisperformed on many transformants derived from REMI transformations, it isprobable that the integration of the plasmid led to short deletions inone or in the two sticky ends of the plasmid BamHI site, thus creatingno BamHI restriction site at the junctions between the plasmid DNA andthe genomic DNA of the transformant. With regard to the digestions withthe enzymes which do not cleave in the sequence of pAN7.1 (ApaI, ClaIand KpnI), the smallest restriction fragment containing the entireplasmid was obtained in the lane corresponding to the KpnI digestion (11kb in size). An additional experiment consisting of hybridization ofSspI restriction fragments of the genomic DNA of the transformant 763with a pUC19 probe was able to show that the sequences of the origin ofreplication of the plasmid in E. coli and of the ampicillin resistancegene were intact.

Example 5 Cloning and Characterization of the Pathogenesis Gene 763

The “plasmid rescue” technique (Timberlake, 1991) was used to clone thegenomic regions located at the point of insertion of the plasmid. Due toits small size, the 11 kb KpnI fragment identified in the molecularanalysis of the mutant was chosen to carry out this experiment (FIG. 2).A restriction analysis of the plasmid DNA of 4 ampicillin-resistantcolonies obtained was performed. A colony bearing the expected plasmid(PRK763) was streaked and multiplied for the purpose of a DNA maxipreparation. An NdeI-SspI genomic DNA fragment of PRK763, 0.4 kb insize, located subsequent to sequencing the regions of genomic origin ofthis plasmid, was used to probe the cosmid library of the strain96/0/76. The cosmids which hybridized with this fragment were isolated(21C7 and 35F3).

A 6 kb XhoI-BglII restriction fragment of the cosmid 35F3 whichhybridizes with the NdeI-SspI restriction fragment of PRK763 was clonedinto the plasmid pCB1265 (FIG. 3). This construct, called pC763 wasintroduced into the genome of the mutant 763 by protoplasttransformation. A pathogenesis assay on detached leaves showed that thephosphinothricin-resistant transformants obtained have the same degreeof virulence as the wild-type strain.

The library of complementary DNAs from the messenger RNAs of genesexpressed in a culture in complete liquid medium was screened with theNdeI-SspI fragment of PRK763. Two types of clone approximately 2 kb longwere recovered. One was shorter than the other by 113 bp at its 5′ end,but 16 bp longer at its 3′ end, this being just before the terminalpolyadenylated sequence. This polyadenylated sequence was present at the3′ ends of the two types of clone isolated. Comparison of this cDNAsequence with that of the corresponding wild-type genomic DNA made itpossible to demonstrate 3 introns of, respectively, 153, 78 and 108 pb.The positions of the translation initiation and termination signals inthe cDNA sequence define an open reading frame 714 pb long. It begins 25bp from the 5′ end of the sequence of the longest cDNA clone and ends1.22 kb from the 3′ end of the sequence of this same clone. This long3′-terminal untranslated sequence comprises many potential terminationsignals in the three possible reading frames.

The search for proteins with sequences homologous to that of P763 wascarried out with the sequence alignment program BLASTP 2.0.8 (Altschulet al., 1997) in all the available databases using the defaultparameters. The only proteins which exhibit a significant degree ofhomology with the pathogenesis protein 763 of Magnaporthe grisea are theputative transcription factor MEAB of Aspergillus nidulans and also thetranscription factors GCN4 and YAP1 of Saccharomyces cerevisiae.

MEAB is thought to be involved in the control of nitrogen assimilationdepending on the nature of the available sources of this element (Polleyand Caddick, FEBS letters 388:200-205, 1996). By virtue of its sequence,MEAB is related to the family of eukaryotic transcription factors of thebZIP type, composed of a dominant basic sequence-specific DNA bindingmotif following by another termed “leucine zipper motif”, required fordimerization of the protein. The degree of similarity between P763 andMEAB is at a maximum in the amino-terminal portion of their sequences,that corresponding to the bZIP domain. Apart from this region, the MEABsequence is longer (400 AA versus 238 in P763) and bears littleresemblance to that of P763 in its carboxy-terminal portion (FIG. 4).

TABLE 4 Search for proteins homologous to the deduced protein of gene763 (Blast P version 2.0.8) % identity and homology at the level of theScore E b-ZIP domain (63 amino acids) 0.00009 38% and 54% MeaB putativetranscription factor of A. nidulans with b-ZIP 0.002 31% and 55% YAP1,transcription factor of S. cerevisae with b-ZIP 0.002 40% and 55% GCN4,transcription factor of S. cerevisae with b-ZIP

The phenotypic analysis of the mutant 763 as a function of its behaviorwith respect to several drugs which interfere with nitrogen metabolismleads to the notion that the gene tagged with the plasmid in the mutant763 is not the equivalent of MEAB in Magnaporthe grisea.

The sequence of the bZIP domain of P763 aligns partially in the samesearch with those of two transcription factors of Saccharomycescerevisiae, GCN4 (protein regulating expression of amino acidbiosynthesis genes; Hinnebusch, PNAS 81:6442-6446,1984) and YAP1(activator of transcription of genes for cellular defense againstoxidative stress; Schnell et al., Curr. Genet. 21 (4-5):269-73 1992),and with the CPC-1 gene of Neurospora crassa (FIG. 5). Genes with asequence homologous to that of the GCN4 gene were identified in thefilamental fungi Neurospora crassa (Paluh et al., PNAS 85 (11)3728-3732, 1988) with the CPC-1 gene and Cryphonectria parasitica (Wanget al., Fungal Genet. Biol. 23 (1):81-94, 1998), and are different fromgene 763 although related.

Example 6 Expression of the Pathogenesis Gene 763

A Northern blot prepared with 10 μg of RNAs extracted from samples ofmycelium grown under several conditions (liquid culture in completemedium or in minimum medium) was hybridized, unsuccessfully, with aprobe corresponding to the sequence of the cDNA of gene 763.

An RT-PCR experiment was carried out with primers located on both sidesof the putative translation termination signal (defined by virtue of theanalysis of the cDNA sequence) and 5 μg of total RNA extracted frommycelium from a liquid culture in complete medium. The amplificationproduct, detected by hybridation with a probe 763, was cloned andsequenced. It shows no differences in size or in sequence with the cDNAclones isolated previously, in particular in the portion correspondingto the untranslated 3′ sequence of the messenger RNA of the gene.

A nested RT-PCR experiment was carried out with 5 μg of RNA frominfected barley leaves extracted 20 hours after inoculation and asecondary amplification was performed with a second pair of internalprimers. An amplification product was detected by hybridization with aprobe 763, revealing expression of this gene during the early steps ofhost colonization (FIGS. 6A and 6B).

1. A method for identifying compounds which inhibit fungal pathogenesis,comprising the following steps: a) bringing a compound into contact witha host organism transformed with a polynucleotide comprising thepromoter of gene 763 and a reporter gene, wherein the sequence of saidpromoter of gene comprises nucleotides 1-705 of SEQ ID NO: 1, andwherein said host organism is a fungus, said host organism expressingsaid reporter gene under the control of said promoter of gene 763; b)detecting the inhibition of the expression of said reporter gene,thereby identifying said compound as a candidate inhibitor of fungalpathogenesis; and c) determining whether the candidate compoundidentified in step (b) inhibits fungal pathogenesis, wherein inhibitionof fungal pathogenesis identifies the compound identified in step (b) asan inhibitor of fungal pathogenesis.